1. Field of the Invention
The present invention relates to a method for transducing neurons with heterologous genes using retrograde viral transport. In greater detail, the present invention relates to a method for introducing and expressing genes in neurons using an adeno-associated virus vector that is capable of retrograde axonal transport. This method has applications in mapping neural pathways, in stimulating or inhibiting the growth of neurons, and in treating various neurodegenerative diseases.
2. Background Art
Diseases of the central nervous system (CNS), more particularly, neurodegenerative diseases, often manifest themselves as a result of the loss or dysfunction of specific projection neurons. Examples include the loss of dopaminergic nigrostriatal projection neurons in Parkinson's disease, entorhinodentate projection neurons in Alzheimer's disease, and spinal motorneurons in amyotrophic lateral sclerosis. See, also, Mark Paul Mattson, Pathogenesis of Neurodegenerative Disorders (Contemporary Neuroscience) (Humana Press; 2001); Marie-Francoise Chesselet (Editor), Molecular Mechanisms of Neurodegenerative Diseases, 1st edition (Humana Press, 2001); Hyman M. Schipper (Editor), Astrocytes in Brain Aging and Neurodegeneration, 1st edition (R G Landes Co., 1998), and the like (each herein incorporated by reference).
Molecular approaches to treat such diseases have opened up new avenues for clinical intervention and assisted scientific study of disease mechanisms. Diseases with a monogenetic etiology may be treated by inserting the gene encoding for the deficient protein. An alternative therapeutic approach for degenerative disorders that may be polygenetic, or that represent the convergence of several risk factors, is to interrupt a common pathway for cell vulnerability. Candidates for this approach include anti-apoptotic genes, such as members of the ras or Bcl-2 family, or molecules such as CGP 3466B, to disrupt the apoptotic cascade and prevent cell death. Neurodegenerative processes are generally characterized by the long-lasting course of neuronal death and the selectivity of the neuronal population or brain structure involved in the lesion. Two main common forms of cell death that have been described in neurons as in other vertebrate tissues i.e., necrosis and apoptosis. Necrosis is the result of cellular “accidents”, such as those occurring in tissues subjected to chemical trauma. The necrotizing cells swell, rupture and provoke an inflammatory response. Apoptosis, on the other hand, is dependent on the cell's “decision” to commit suicide and die, and therefore is referred to as “programmed cell death” (PCD). The course of apoptotic death is characterized by a massive morphological change, including cell shrinkage, nuclear (chromosome) condensation and DNA degradation. Activation of PCD in an individual cell is based on its own internal metabolism, environment, developmental background and its genetic information. Such a situation occurs in most of the neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases and amyotrophic lateral sclerosis (ALS). In these pathological situations, specific neurons undergo apoptotic cell death characterized by DNA fragmentation, increased levels of pro-apoptotic genes and “apoptotic proteins” both, in human brain and in experimental models. It is of utmost importance to conclusively determine the mode of cell death in neurodegenerative diseases, because new “anti-apoptotic” compounds may offer a means of protecting neurons from cell death and of slowing the rate of cell degeneration and illness progression (see, e.g., Offen et al., J Neural Transm Suppl. (2000) 58:153–66).
However, one problem involved in using such in vivo genetic approaches to effectively treat diseases of the CNS is that the CNS has a heterogeneous cytoarchitecture. For example, intracranial injection must deliver the vector to a specific location without damaging the targeted cells or causing collateral infection of nearby cells. This precision of delivery is difficult to achieve since many target neuronal populations are physically intermixed with many different neurons. In addition, many target neuronal populations are effectively inaccessible using current delivery methods.
The development of the adeno-associated virus (AAV) vector, which is capable of infecting post-mitotic neurons, has greatly facilitated in vivo gene delivery to the central nervous system (CNS). Gene therapy vectors based on the adeno-associated virus (AAV) are being developed for a widening variety of therapeutic applications. Enthusiasm for AAV is due, not only to the relative safety of these vectors, but also to advances in understanding of the unique biology of this virus. Wild-type AAV is a nonpathogenic parvovirus that is incapable of replication without the co-infection of a helper virus, such as adenovirus or HSV. When used as a vector, almost all of the AAV genome is deleted, leaving only terminal repeats with DNA replication and packaging signals. This removal of the viral coding sequence prevents the generation of wild-type helper virus and reduces the possibility of immune reactions caused by undesired viral gene expression. It is also possible to remove populations of contaminating helper virus from the AAV vector population on the basis of structurally distinct coat proteins.
AAV is capable of transducing both dividing and non-dividing cells, and is thus of particular applicability to the CNS, where most of the cells are nondividing. AAV is an integrating virus and the integrated provirus is very stable, thus offering long-term transduction; there have been reports of stable gene expression for up to two years after AAV-mediated gene delivery. Lastly, in contrast to other viral vectors such as adenovirus, which induce severe immune responses, AAV vectors characteristically exhibit no cellular immune response against vector-transduced cells. In sum, AAV is particularly attractive due to its low toxicity and immunogenicity and its long-term gene expression.
AAV vectors were previously thought to be of use only in the transduction of local neurons and anterograde axon tracing. For example, Chamberlin et al. found that in contrast to other viral vectors that may be retrogradely transported by neurons in remote regions of the brain, the AAV appeared to transduce only local neurons (N. Chamberlin et al., Brain Research 793:174 (1998)). In addition to AAV, DeFalco et al. report the use of the pseudorabies virus to trace the locations of selected neurons (DeFalco et al., Virus-Assisted Mapping of Neural Inputs to a Feeding Center in the Hypothalamus, Science 291:2608–2613 (March 2001)).
Such retrograde transport has been reported for herpes simplex (HSV), adenovirus, and pseudorabies virus. However, viral toxicity from these vector systems can limit expression duration and yield variable results. Viruses used as vectors in this manner generally carry deletions in viral genes, either in order to limit viral toxicity or to make the viruses unable to replicate. However, although some viral genes are inactivated in these vectors, many functional viral genes are present, which may make the vectors toxic or reactivate latent viruses in recipient cells. Even so-called “defective viral vectors,” which have been produced from HSV and the like and which contain no viral genes at all, still present problems with continued gene expression, pathogenicity, and reversion to wild type. Additionally, such vectors, such as adenovirus vectors, often have capsid proteins that provoke a strong immune response. To date, retrograde axonal transport has not been accomplished with a substantially nontoxic genetic vector. As used herein, what is meant by a “substantially nontoxic genetic vector” is a vector which is not directly toxic to transfected cells and which does not cause a post-transfection immune response. Such a substantially non-toxic, retrograde vector delivery system would be of great use in the mapping of CNS circuitry and in the treatment of the various neurodegenerative illnesses described above.
Thus, what is needed in the art is an efficient mechanism for delivering heterologous genes to neuronal cells. Tackling neurodegenerative diseases represents a formidable challenge for our ageing society. Recently, major achievements have been made in understanding the molecular mechanisms responsible for such diseases, and, simultaneously, numerous proteins such as neurotrophic factors, anti-apoptotic or anti-oxidant have been identified as potential therapeutic agents. Although many neurotrophic factors have been tested on individuals suffering from various neurodegenerative disorders, to date none has shown efficacy. Inadequate protein delivery is believed to be part of the problem. Recent improvements in pump technology, as well as in cell and gene therapy, are providing innovative ways to allow localized, regulatable delivery of proteins in brain parenchyma, opening new avenues for clinical trials in the not so distant future (see, e.g., Aebischer & Ridet, Trends Neurosci. 2001 24(9):533–40 (generally describing therapeutic genes and methods, incorporated by reference herein). The invention described herein provides such a mechanism.